Stabilization of the tertiary structure of yeast phenylalanine tRNA by [Co(NH3)6]3+: X-ray evidence for hydrogen bonding to pairs of guanine bases in the major groove

The sites of three [Co(NH₃)₆]³⁺ ions bound to the phenylalanine tRNA of yeast have been determined by X-ray diffraction analysis. [Co(NH₃)₆]³⁺ binds to purine-purine sequences in yeast tRNA^Phe. It is different from the binding of Co²⁺, which binds to the base and phosphate of residue G15. There are no direct metal-nucleotide bonds, although hydrogen bonding of the coordinated ammines to double-helical guanylguanosine sequences in the major groove and to phosphate oxygens in neighboring polynucleotide strands increases the stability of the structure. Hydrogen-bonding appears to be via cis ammine ligands to N(7) and O(6) positions of adjacent purine bases.     

Effect of 5-amino-4-imidazolecarboxamide riboside on renal ammoniagenesis. Study with [15N]aspartate

[15N]Aspartate and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) were used to evaluate the contribution of the purine nucleotide cycle to ammonia production in renal tubules isolated from control and chronically acidotic rats. Addition of 1 mM AICAriboside to incubation medium containing 2.5 mM [15N] aspartate significantly stimulated production of 15NH3 and 15N in the 6-amino group of adenine nucleotides during a 30-min incubation. In tubules from both control and acidotic animals, the levels of ATP, AMP, and NH3 were increased. In contrast, 5 mM AICAriboside inhibited 15NH3 production and reduced the total purine nucleotide content. In tubules from acidotic rats, enrichment in 15NH3 exceeded that in the 6-amino group of the adenine nucleotides, indicating that no precursor-product relationship existed between the purine nucleotide cycle and ammonia. Conversely, in tubules from control rats, 15N enrichment in the 6-amino group of the adenine nucleotides exceeded that in NH3. This relationship obtained whether or not AICAriboside was included in the incubation mixture. The current investigations show that the metabolism of aspartate through the purine nucleotide cycle is lower in renal tubules obtained from chronically acidotic rats than in control tubules. The observations indicate that AICAriboside has a biphasic effect on renal ammoniagenesis and adenine nucleotide synthesis, and suggest a possible clinical use of AICAriboside in cases of impaired ammonia formation in renal failure.     

Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison

Related domains containing the purine NTP-binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N-terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N-ligase-kinase-phosphohydrolase-C, whereas polynucleotide kinase and CNPase encompass only the two C-terminal domains in the same order.     

The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices.

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.     

The synthesis and properties of N6-substituted 2-amino-purine derivatives.

1. The synthesis, ultraviolet absorption spectra, and behaviour in alkali of N6-methoxy-, N6-methyl, hydroxy-, and N6-hydroxy-2-aminopurines have been described 2. N6-Methoxy-2-aminopurine riboside 5'-pyrophosphate has been prepared and used for polymerization with polynucleotide phosphorylase. 3. The copolymer containing N6-methoxy-2-aminopurine riboside and adenosine residues has been obtained; attempts to synthesize the homopolymer have not been successful. 4. All the purine analogues synthesized have been tested and shown to act mutagenically on Salmonella typhimurium TA1530.     

Evidence for Ammonium-dependent de Novo Synthesis of Glutamate Dehydrogenase in Detached Oat Leaves

Glutamate dehydrogenase becomes density labeled through the incorporation of deuterium and (15)N when detached oat leaves (Avena sativa var. Fulghum) are incubated in the presence of ammonia. The enzyme has been isolated by means of DEAE-cellulose chromatography, ammonium sulfate precipitation, isopycnic equilibrium centrifugation, and disc electrophoresis from leaves fed l-methionine-(35)S. Radioactivity is incorporated into isozyme 1 of glutamate dehydrogenase, whereas isozyme 2, detected only in the absence of ammonia, has not been labeled. Cycloheximide, chloramphenicol, puromycin, and 6-methyl purine inhibit the elevation of glutamate dehydrogenase by ammonia. It is suggested that the increase in glutamate dehydrogenase activity is due to de novo synthesis of isozyme 1.     

Interaction of transition metal ions with Z form poly d(A-C).poly d(G-T) and poly d(A-T) studied by I.R. spectroscopy.

Interactions between Ni2+, Co2+ and purine bases have been studied by I.R. spectroscopy in the case of double stranded regularly alternating purine-pyrimidine polynucleotides poly d(A-T), poly d(A-C).poly d(G-T) and poly d(G-C). The spectra of polynucleotide films have been recorded in hydration and salt content conditions which correspond to the obtention of the classical right-handed (A,B) and left-handed (Z) helical conformations. Selective deuteration of the 8C site of purines has been obtained and is used to detect interactions between the transition metal ions and the adenine or guanine bases. The spectral region between 1500 and 1250 cm-1 corresponding to base in-plane vibrations and involving also the glycosidic linkage torsion is discussed in detail. The selective interaction between the transition metal ion and the 7N site of the purine base is considered to be partly responsible for the stabilization of the base in a syn conformation, which favours the adoption by the polynucleotide (poly d(G-C), poly d(A-C).poly d(G-T) or poly d(A-T)) of a Z type conformation.     

The purine nucleotide cycle and ammoniagenesis in rat kidney tubules

The contribution of the purine nucleotide cycle to renal ammoniagenesis was examined in cortical tubule suspensions prepared from acidotic rats and incubated with [alpha-15N]glutamine, [15N]glutamate, or [15N]aspartate. Labeling of ammonia and adenine nucleotides was determined after enzymatic transformations designed to circumvent the technical problem that 15NH3 and H2O have the same nominal mass. Labeling of the adenine nucleotide was undetectable (less than 10%) even after 1 h of incubation. From the measured concentrations of adenine nucleotides and ammonia and the labeling of the ammonia, the flux through the purine nucleotide cycle was calculated to account for less than 1% of the deamination of alpha-amino groups from all three substrates. The glutamate dehydrogenase reaction is therefore the likely pathway for deamination. The rate of 15NH3 production from [alpha-15N]glutamine was two or three times greater than from added [15N]glutamate, indicating a preference for intracellularly generated glutamate. 15NH3 production from added [15N]aspartate was similar to and perhaps slightly greater than that from added [15N]glutamate.     

Procedure for C2 deuteration of nucleic acids and determination of A psi 31 pseudouridine conformation by nuclear Overhauser effect in yeast tRNAPhe

Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA. Deuterated tri-ethyl orthoformate was condensed with 5(4)-amino imidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine. C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated yeast tRNAPhe was isolated. This C2 deuterated tRNAPhe was used to identify A psi 31 and U8-A14. A psi 31 was found to be bonded through N1H. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.     

Alanine metabolism in the perfused rat liver. Studies with (15)N.

We have utilized [(15)N]alanine or (15)NH(3) as metabolic tracers in order to identify sources of nitrogen for hepatic ureagenesis in a liver perfusion system. Studies were done in the presence and absence of physiologic concentrations of portal venous ammonia in order to test the hypothesis that, when the NH(4)(+):aspartate ratio is >1, increased hepatic proteolysis provides cytoplasmic aspartate in order to support ureagenesis. When 1 mm [(15)N]alanine was the sole nitrogen source, the amino group was incorporated into both nitrogens of urea and both nitrogens of glutamine. However, when studies were done with 1 mm alanine and 0.3 mm NH(4)Cl, alanine failed to provide aspartate at a rate that would have detoxified all administered ammonia. Under these circumstances, the presence of ammonia at a physiologic concentration stimulated hepatic proteolysis. In perfusions with alanine alone, approximately 400 nmol of nitrogen/min/g liver was needed to satisfy the balance between nitrogen intake and nitrogen output. When the model included alanine and NH(4)Cl, 1000 nmol of nitrogen/min/g liver were formed from an intra-hepatic source, presumably proteolysis. In this manner, the internal pool provided the cytoplasmic aspartate that allowed the liver to dispose of mitochondrial carbamyl phosphate that was rapidly produced from external ammonia. This information may be relevant to those clinical situations (renal failure, cirrhosis, starvation, low protein diet, and malignancy) when portal venous NH(4)(+) greatly exceeds the concentration of aspartate. Under these circumstances, the liver must summon internal pools of protein in order to accommodate the ammonia burden.     

Expression and purification of imidazole glycerol phosphate synthase from Saccharomyces cerevisiae

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N(1)-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-car boxamide ribonucleotide (PRFAR). This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis. The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IGP synthase established that this protein is bifunctional, representing a fusion between the N-terminal HisH domain and a C-terminal HisF domain. Catalytically active yeast HIS7 was expressed in a bacterial system under the control of T7 polymerase promoter. The recombinant enzyme was purified to homogeneity and the native molecular weight and steady-state kinetic constants were determined. The yeast enzyme is distinguished from the Escherichia coli IGP synthase in its utilization of ammonia as a substrate. HIS7 displays a higher K(m) for glutamine and a lower turnover in the ammonia-dependent IGP synthase activity. As observed with the E. coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in the presence of a nucleotide substrate or analog. The purification and characterization of the S. cerevisiae enzyme will enable a more detailed investigation of the biochemical mechanisms that mediate the ammonia-transfer process. The fused structural feature of the HIS7 protein and the development of a high-level production system for the active enzyme elevate the potential for determination of its three-dimensional structure through X-ray crystallography.     

Enzyme concentration affects the allosteric behavior of yeast phosphofructokinase

The influence of enzyme concentration on the kinetic behavior of yeast phosphofructokinase has been examined. A marked decrease in the ATP inhibition was observed when the enzyme activity was studied in permeabilized cells (in situ) as well as when the kinetic study was carried out with the purified yeast enzyme at a concentration of 120 micrograms/ml as compared to a 100-fold diluted enzyme. A similar result was obtained by adding polyethylene glycol either to a cell free extract or to the diluted pure enzyme to increase the local protein concentration. However, enzyme concentration had no significant effect on the fructose-6-P saturation curve. These results provide evidence that the allosteric behavior of yeast phosphofructokinase is affected by enzyme concentration.     

Early events in the initiation of ammonia formation in kidney

Experiments were designed to examine the early events in the initiation of glutamate deamination in kidney. Perfused kidneys from methionine sulfoximine-treated rats formed ammonia from [15N]glutamate via the purine nucleotide cycle. The turnover of the 6-amino group of adenine nucleotides to yield ammonia occurred at the rate of 0.30 mumol/g of kidney/min. This rate is 3-4 times larger than in liver and is in agreement with published rates of the purine nucleotide cycle in kidney. The addition of 0.1 mM fluorocitrate to glutamate perfusions stimulated ammonia formation 3 1/2-fold. The turnover of the 6-amino group of adenine nucleotides increased during the first 5 min after adding fluorocitrate to form ammonia predominately from tissue glutamate and aspartate. This turnover correlates with a 3 1/2-fold increase in kidney tissue IMP levels. As the ATP/ADP ratio fell the purine nucleotide cycle was inhibited and glutamate dehydrogenase was stimulated to form ammonia stoichiometric with glutamate taken up from the perfusate. Ammonia formation via glutamate dehydrogenase occurred at a rate of 1.0 mumol/g of kidney/min. Fluorocitrate completely blocked ammonia formation from aspartate in perfusions. The perfused kidney formed ammonia from aspartate via the purine nucleotide cycle at a rate of 1.0 mumol/g of kidney/min. The results indicate a discrete role for aspartate in renal metabolism. Ammonia formation via the purine nucleotide cycle can occur at significant rates and equal to the rate of ammonia formation from glutamate via glutamate dehydrogenase.     

Effect of High-Dose Nano-selenium and Selenium–Yeast on Feed Digestibility, Rumen Fermentation, and Purine Derivatives in Sheep

The aim of this study was to evaluate the effect of nano-selenium (NS) and yeast?Cselenium (YS) supplementation on feed digestibility, rumen fermentation, and urinary purine derivatives in sheep. Six male ruminally cannulated sheep, average 43.32?±?4.8?kg of BW, were used in a replicated 3?×?3 Latin square experiment. The treatments were control (without NS and YS), NS with 4?g nano-Se (provide 4?mg Se), and YS with 4?g Se-yeast (provide 4?mg Se) per kilogram of diet dry matter (DM), respectively. Experimental periods were 25?days with 15?days of adaptation and 10?days of sampling. Ruminal pH, ammonia N concentration, molar proportion of propionate, and ratio of acetate to propionate were decreased (P?<?0.01), and total ruminal VFA concentration was increased with NS and YS supplementation (P?<?0.01). In situ ruminal neutral detergent fiber (aNDF) degradation of Leymus chinensis (P?<?0.01) and crude protein (CP) of soybean meal (P?<?0.01) were significantly improved by Se supplementation. Digestibilities of DM, organic matter, crude protein, ether extract, aNDF, and ADF in the total tract and urinary excretion of purine derivatives were also affected by feeding Se supplementation diets (P?<?0.01). Ruminal fermentation was improved by feeding NS, and feed conversion efficiency was also increased compared with YS (P?<?0.01). We concluded that nano-Se can be used as a preferentially available selenium source in ruminant nutrition.     

A comparison of the estimates of whole-body protein turnover in parenterally fed neonates obtained using three different end products

Protein turnover rates in neonates have been calculated largely by measuring urinary [15N]urea enrichment following administration of [15N]glycine. Although ammonia has been increasingly recognized as an end product of nitrogen metabolism, in neonates it yields a different estimate of protein turnover than does urea. Comparisons of ammonia and urea end products in parenterally fed neonates have not previously been reported. A third and independent way of estimating protein turnover, developed for adults, is to use breath 13CO2 as an end product following administration of [1-13C]leucine. We therefore carried out simultaneous measurements of protein turnover in 10 parenterally fed neonates, using the three end products. The infants were clinically stable, weighed 2.6 +/- 0.2 kg, and received 3.1 +/- 0.2 g.kg-1.d-1 of amino acid, 2.2 +/- 0.1 g.kg-1.d-1 of lipids, and an energy intake of 90 +/- 4 kcal.kg-1.d-1 (1 kcal = 4.186 kJ). The turnover estimates derived from the 13CO2 and [15N]urea end products were very similar. The [15N]ammonia end product produced values approximately 66% (p less than 0.01) of the other two. We conclude that the ammonia and urea end products probably originate in different precursor pools. The similarity of the urea and breath carbon dioxide results helps validate the use of the urea end product in studying the nitrogen metabolism of parenterally fed neonates. Ideally in future studies two or more end products should be used, since they provide information about different aspects of the neonates' protein metabolism.     

Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction.

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3.     

Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes

When mammalian cells are grown in medium containing [3H]spermidine, a single major tritiated protein identical to eukaryotic initiation factor 4D becomes labeled. This protein contains 1 residue/molecule of tritiated hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), a rare amino acid which has been found in no other protein. In order to investigate the conservation of this protein, we examined two nonmammalian eukaryotes, the yeast Saccharomyces cerevisiae and the insect Drosophila melanogaster, and the eubacterial prokaryote Escherichia coli for the presence of the hypusine-containing protein. When the eukaryotic cells were grown in the presence of [3H]spermidine, electrophoretic analysis revealed a single labeled protein. In each case, the apparent molecular weight was near 18,000 and the relative pI was approximately 5.2, similar to the hypusine-containing protein of mammals. Amino acid analysis confirmed the presence of tritiated hypusine in each case, and silver staining of two-dimensional polyacrylamide gels demonstrated that, in yeast and fruit flies as in mammals, the protein is relatively abundant. In the eubacterium E. coli, one tritiated protein was predominant, but its molecular weight was 24,000 and we found no evidence that it contained tritiated hypusine. We found no evidence for the existence of the hypusine-containing protein in the archaebacterium Methanococcus voltae. These data suggest that the hypusine-containing protein is conserved among eukaryotes.